Murine Wee1 plays a critical role in cell cycle regulation and pre-implantation stages of embryonic development

Wee1 kinase regulates the G2/M cell cycle checkpoint by phosphorylating and inactivating the mitotic cyclin-dependent kinase 1 (Cdk1). Loss of Wee1 in many systems, including yeast and drosophila, leads to premature mitotic entry. However, the developmental role of Wee1 in mammals remains unclear. In this study, we established Wee1 knockout mice by gene targeting. We found that Wee-/- embryos were defective in the G2/M cell cycle checkpoint induced by gamma-irradiation and died of apoptosis before embryonic (E) day 3.5. To study the function of Wee1 further, we have developed MEF cells in which Wee1 is disrupted by a tamoxifen inducible Cre-LoxP approach. We found that acute deletion of Wee1 resulted in profound growth defects and cell death. Wee1 deficient cells displayed chromosome aneuploidy and DNA damage as revealed by gamma-H2AX foci formation and Chk2 activation. Further studies revealed a conserved mechanism of Wee1 in regulating mitotic entry and the G2/M checkpoint compared with other lower organisms. These data provide in vivo evidence that mammalian Wee1 plays a critical role in maintaining genome integrity and is essential for embryonic survival at the pre-implantation stage of mouse development.     

Responses to climate by tree-ring widths and maximum latewood densities of two Abies species at upper and lower altitudinal distribution limits in central Japan

This study examined the effects of climate on tree-ring widths and maximum latewood densities of Abies veitchii and Abies mariesii at the upper and lower distribution limits in central Japan. A. veitchii and A. mariesii dominated at the lower and upper parts of the subalpine zone, respectively. Residual chronologies of tree-ring width and maximum latewood density were developed for the two Abies species at the upper and lower distribution limits, and were compared with monthly mean temperatures and monthly sums of precipitation. Tree-ring widths of the two Abies species at the upper and lower distribution limits positively correlated with temperatures during the beginning of the dormant season and during the growing season of the current year, except for A. veitchii at the lower distribution limit, which showed no positive correlation with temperature. Maximum latewood densities of the two Abies species at the upper and lower distribution limits positively and negatively correlated with temperatures and precipitation, respectively, during the growing season of the current year. Therefore, tree-ring widths and maximum latewood densities of the two Abies species were sensitive to low temperature, except for the tree-ring width of A. veitchii at the lower distribution limit with the warmest thermal conditions along the altitude. Global warming is suggested to affect maximum latewood densities and tree-ring widths of the two Abies species along the altitude.     

The F-BAR Protein Rapostlin Regulates Dendritic Spine Formation in Hippocampal Neurons

Pombe Cdc15 homology proteins, characterized by Fer/CIP4 homology Bin-Amphiphysin-Rvs/extended Fer/CIP4 homology (F-BAR/EFC) domains with membrane invaginating property, play critical roles in a variety of membrane reorganization processes. Among them, Rapostlin/formin-binding protein 17 (FBP17) has attracted increasing attention as a critical coordinator of endocytosis. Here we found that Rapostlin was expressed in the developing rat brain, including the hippocampus, in late developmental stages when accelerated dendritic spine formation and maturation occur. In primary cultured rat hippocampal neurons, knockdown of Rapostlin by shRNA or overexpression of Rapostlin-QQ, an F-BAR domain mutant of Rapostlin that has no ability to induce membrane invagination, led to a significant decrease in spine density. Expression of shRNA-resistant wild-type Rapostlin effectively restored spine density in Rapostlin knockdown neurons, whereas expression of Rapostlin deletion mutants lacking the protein kinase C-related kinase homology region 1 (HR1) or Src homology 3 (SH3) domain did not. In addition, knockdown of Rapostlin or overexpression of Rapostlin-QQ reduced the uptake of transferrin in hippocampal neurons. Knockdown of Rnd2, which binds to the HR1 domain of Rapostlin, also reduced spine density and the transferrin uptake. These results suggest that Rapostlin and Rnd2 cooperatively regulate spine density. Indeed, Rnd2 enhanced the Rapostlin-induced tubular membrane invagination. We conclude that the F-BAR protein Rapostlin, whose activity is regulated by Rnd2, plays a key role in spine formation through the regulation of membrane dynamics.     

Extended expression of liver functions of hepatocytes in collagen-contained cell aggregates (cell packs)

The functions of hepatocytes under the collagen-contained cell aggregate (cell pack) conditions were studied using liver-specific protein synthesis. Freshly isolated murine hepatocytes were suspended in the medium containing collagen and centrifuged, and the resultant cell masses were cultured on the porous membranes floating on the medium. In these cultures cells were attached to each other three-dimensionally with collagen present in the intercellular spaces. Cultured hepatocytes in the cell pack maintained high and stable activity in the expression of their functions for more than 2 weeks, even when cultured with the medium lacking any hormones and serum, whereas hepatocytes in monolayer cultures lost their functions within a week.Similarly, when the cell packs of rat hepatocytes were transplanted into rat spleens, they could retain viability in the form of cell aggregate with the expression of liver-specific albumin mRNA at a higher level than in the transplantated cell suspensions.The lifespan and the initial expression level of hepatocellular functions inculture were similar to that of the cell pack in cell aggregates without collagen and in cellular monolayers on the collagen gel respectively.It was concluded that the condition where cells are in contact witheach other has an important role in the expression of hepatocellular functions and collagen present in the intercellular spaces enhances the functional levels.     

A Role for Tissue Factor in Cell Adhesion and Migration Mediated by Interaction with Actin-binding Protein 280

Tissue factor (TF), the protease receptor initiating the coagulation system, functions in vascular development, angiogenesis, and tumor cell metastasis by poorly defined molecular mechanisms. We demonstrate that immobilized ligands for TF specifically support cell adhesion, migration, spreading, and intracellular signaling, which are not inhibited by RGD peptides. Two-hybrid screening identified actin-binding protein 280 (ABP-280) as ligand for the TF cytoplasmic domain. Extracellular ligation of TF is necessary for ABP-280 binding. ABP-280 recruitment to TF adhesion contacts is associated with reorganization of actin filaments, but cytoskeletal adaptor molecules typically found in integrin-mediated focal contacts are not associated with TF. Chimeric molecules of the TF cytoplasmic domain and an unrelated extracellular domain support cell spreading and migration, demonstrating that the extracellular domain of TF is not involved in the recruitment of accessory molecules that influence adhesive functions. Replacement of TF's cytoplasmic Ser residues with Asp to mimic phosphorylation enhances the interaction with ABP-280, whereas Ala mutations abolish coprecipitation of ABP-280 with immobilized TF cytoplasmic domain, and severely reduce cell spreading. The specific interaction of the TF cytoplasmic domain with ABP-280 provides a molecular pathway by which TF supports tumor cell metastasis and vascular remodeling.     

Herpes simplex virus UL14 protein blocks apoptosis

We report that an HSV-2 UL14 protein expressing cell line (14/HEp-2) was more resistant to apoptosis induced by osmotic shock and certain drugs than its parental cell line. Furthermore, HSV-1 UL14 protein deletion virus (UL14D) showed weaker inhibition of apoptosis compared to the rescued virus UL14R. The protein's anti-apoptotic function may derive from its heat shock protein-like properties.     

Development of On-Chip Multi-Imaging Flow Cytometry for Identification of Imaging Biomarkers of Clustered Circulating Tumor Cells

An on-chip multi-imaging flow cytometry system has been developed to obtain morphometric parameters of cell clusters such as cell number, perimeter, total cross-sectional area, number of nuclei and size of clusters as “imaging biomarkers”, with simultaneous acquisition and analysis of both bright-field (BF) and fluorescent (FL) images at 200 frames per second (fps); by using this system, we examined the effectiveness of using imaging biomarkers for the identification of clustered circulating tumor cells (CTCs). Sample blood of rats in which a prostate cancer cell line (MAT-LyLu) had been pre-implanted was applied to a microchannel on a disposable microchip after staining the nuclei using fluorescent dye for their visualization, and the acquired images were measured and compared with those of healthy rats. In terms of the results, clustered cells having (1) cell area larger than 200 µm² and (2) nucleus area larger than 90 µm² were specifically observed in cancer cell-implanted blood, but were not observed in healthy rats. In addition, (3) clusters having more than 3 nuclei were specific for cancer-implanted blood and (4) a ratio between the actual perimeter and the perimeter calculated from the obtained area, which reflects a shape distorted from ideal roundness, of less than 0.90 was specific for all clusters having more than 3 nuclei and was also specific for cancer-implanted blood. The collected clusters larger than 300 µm² were examined by quantitative gene copy number assay, and were identified as being CTCs. These results indicate the usefulness of the imaging biomarkers for characterizing clusters, and all of the four examined imaging biomarkers—cluster area, nuclei area, nuclei number, and ratio of perimeter—can identify clustered CTCs in blood with the same level of preciseness using multi-imaging cytometry.     

A novel microperoxidase activity: methyl viologen-linked nitrite reducing activity of microperoxidase

To investigate the nitrite reducing activity of microperoxidases (mps) in the presence of methyl viologen and dithionite, the fragments C14-K22 (mp9), V11-L32 (mp22), and G1-M65 (mp65) containing heme were prepared by enzymatic hydrolysis of commercially equine heart cytochrome c (Cyt c), in which His is axially coordinated to heme iron, and acts as its fifth ligand. The nitrite reducing activity of mps was measured under anaerobic condition, and the nitrite reducing activity of mps increased with the cutting of the peptide chain. The activity of the shortest nonapeptide mp9 was approximately 120-fold that of Cyt c (104 amino acid residues) and 3.2-fold that of nitrite reductase (EC 1.7.7.1) from Escherichia coli. In the nitrite reduction by mp, nitrite was completely reduced to ammonia. We presumed that ferrous mps reduced NO2- to NO by donating one electron, the NO was completely reduced to NH4+ under anaerobic condition via ferrous-NO complexes as a reaction intermediate using visible spectra and ESR spectra, and this overall reaction was a 6-electron and 8-proton reduction. Sepharose-immobilized mp9 had a nitrite reducing activity similar to that of mp9 in solution, and the resin retained the activity after five uses and even 1-year storage. The mp will be able to use as a substitute for nitrite reductase.     

Cancer chemopreventive activity of serratane-type triterpenoids from Picea jezoensis

To search for cancer chemopreventive agents from natural sources, 13alpha,14alpha-epoxy-21alpha-methoxyserratan-3-one, 21alpha-methoxyserrat-13-en-3-one, and 21alpha-hydroxy-3beta-methoxyserrat-14-en-30-al isolated from the cuticle of Picea jezoensis (Sieb. et Zucc.) Carr. var. jezoensis (Pinaceae) were investigated for inhibitory effects in a two-stage mouse skin carcinogenesis test on mouse skin with 7,12-dimethylbenz[a]anthracene (DMBA) as initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as promoter. 21Alpha-hydroxy-3beta-methoxyserrat-14-en-30-al and 13alpha,14alpha-epoxy-21alpha-methoxyserratan-3-one were found to exhibit strong antitumor-promoting activities in the in vivo carcinogenesis test.